Rapid Synthesis of a Natural Product-Inspired Uridine Containing Library

Wei-Chieh Cheng,* Wan-Ju Liu, Kung-Hsiang Hu, Yee-Ling Tan, Yan-Ting Lin, Wei-An Chen,

Research Article

Rapid Synthesis of a Natural Product-Inspired Uridine Containing

Library

and Lee-Chiang Lo *

Cite This: https://dx.doi.org/10.1021/acscombsci.0c00011

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sı Supporting Information

ABSTRACT: The preparation of natural product-inspired nucleo-

side analogs using solution-phase parallel synthesis is described.

The key intermediates containing alkyne and N-protected amino

moieties were developed to allow for further skeleton and

substituent diversity using click chemistry and urea or amide

bond formation. Rapid puriﬁcation was accomplished using solid-

phase extraction. The obtained library comprised 80 molecules

incorporating two diversity positions and one chiral center, each of

which was eﬃciently prepared in good purity and acceptable

overall yield. A bacterial morphology study was also performed.

KEYWORDS: combinatorial chemistry, natural product-inspired nucleosides, chemical space, semiautomated synthesis,

uridine-containing library

INTRODUCTION

diversity positions and synthesized by way of ﬂexible,

■

fragment-assembly strategies have not been investigated.

Our research interests include the design and synthesis of

Natural products are an important source of inspiration in drug

discovery, medicinal chemistry, and chemical biology.

Among them, uridine-containing natural products show a

wide range of bioactivities, such as anticancer, antifungal,

antiviral, and antibiotic activity, and are implicated in several

−3

biologically interesting natural product-inspired scaﬀolds and

libraries via divergent synthesis or combinatorial chemis-

19−22

try.

Recently, we reported the systematic preparation of

−6

bacterial Park nucleotide-based molecules, one class of UDP-

nucleoside metabolic pathways. For example (Figure 1), the

polyoxins, a group of antifungal nucleosides that inhibit chitin

synthase, were isolated from Streptomyces cacaoi subsp.

type natural products, for an enzyme substrate speciﬁcity

study. Inspired by naturally occurring uridine-containing

molecules, nucleoside antibiotics, and uridine metabolic

pathways, we realized that diverse chemical synthesis of

these uridine-containing scaﬀolds, molecules, or libraries is

needed to increase our chemical space coverage and for their

further study in forward or reverse chemical genetic screen-

7,8

Asoensis; the muraymycins were isolated from a culture

broth of Streptomyces sp. and found to show excellent

−12

antimicrobial activity against Gram-positive bacteria;

and

tunicamycin, produced by Streptomyces lysosuperficus, is a

nucleoside antibiotic complex under investigation for its

inhibition of polyisoprenyl-phosphate N-acetylhexosamine-1-

phosphate transferases (PNPT) with view to the development

of a novel anticancer drug.

These uridine-containing natural products are all biologically

relevant molecules and share a common structural feature (the

24−27

ing.

Here, we report an eﬃcient, solution-phase parallel synthetic

approach to prepare a diverse uridine-containing library by

assembly of multifragments or substituents within a single

framework (two diversity positions, three conjugations link-

ages, one space, and one chiral center) under mild reaction

3,14

′-substituted uridine moiety) but are otherwise structurally

diverse and complex. Thus, it is diﬃcult to clearly deﬁne their

real core structures or diversity positions. Also, their structural

complexity (in particular, the large number of stereogenic

centers) and very low natural availability hamper their further

study. To date, only straightforward one-dimensional chemical

diversity of 5′-substituted uridines has been extensively

Received: January 23, 2020

Revised: August 12, 2020

Published: August 24, 2020

5−17

reported;

to the best of our knowledge, more structurally

diverse uridine-based libraries incorporating more than two

XXXX American Chemical Society

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Research Article

highly moisture sensitive and not convenient. Therefore, we

decide to utilize an amide or urea moiety as our linkage III

instead of the glycosidic linkage to eﬃciently perform the

substituent diversity. In addition, the triazole formation

(

CuAAC) and the amide bond formation will be applied as

our linkage I and II, respectively (Scheme 1). Several ﬂexible

and semiautomated equipment, including a solution-phase

synthesizer, multichannel liquid handler, and centrifugal

evaporator were used to accelerate the preparation process.

Product puriﬁcation was rapidly accomplished using solid-

32−34

phase extraction.

RESULTS AND DISCUSSION

Design and Retrosynthetic Analysis. Scheme 2 depicts

our retrosynthetic analysis. The general structure of target

■

Scheme 2. Retrosynthetic Analysis of 5′-Triazole-

Substituted Uridines

Figure 1. Examples of bioactive molecules containing a uridine

moiety.

conditions. Scheme 1 is a schematic depiction of our synthetic

strategy. Several ﬂexible and convenient bioconjugation

methods, such as Cu-catalyzed azide−alkyne cycloaddition

8,29

(

CuAAC),

amide bond formation, and urea bond

library I includes two structurally diverse fragments A and B

and one position of conﬁguration diversity (at the C5′

position). Fragment A (see in Scheme 1) incorporates several

structural features including the triazole-based linkage I, the

amide-based linkage II, a ﬂexible spacer, and a lipophilic

moiety. In contrast, the amino group attached at the C5′

position will be used for the attachment of fragment B through

formation, will be applied in this study. Notably, some

natural products or bioactive compounds (see in Figure 1)

possess a glycosidic linkage, but this formation condition is

Scheme 1. Principle of Our Structural Design for Uridine-

Based Chemical Space

(

thio-) urea or amide bond formation with various isocyanates

or carboxylic acids. Scaﬀold II will be prepared from the key

intermediate III, derived from uridine.

Synthesis of Key Intermediates 5S and 5R. As shown in

Scheme 3, alkyne alcohol 1R was prepared from uridine as

previously described, with minor modiﬁcations. Mesylation

of 2R, followed by S 2 substitution, with sodium azide in

DMF at 50 °C gave 4S in 84% overall yield over two steps.

Reduction of azide 4S under Staudinger conditions (PMe₃/

H O/THF) followed by N-Boc protection gave the ﬁrst N-

protected amino alkyne 5S in 63% yield (two steps). N-

protected amino alkyne 5R was accessible from 1R by either

Mitsunobu reaction, mesylation, and Boc-protection or via an

intramolecular Mitsunobu cyclization to give cyclized inter-

mediate 7, followed by ring opening with sodium azide.

Notably, oxidation of 1R , followed by reduction (see

Supporting Information (SI)) did not give us a better

diastereoselective ratio for a separation beneﬁt (approximately

:1). We also found that use of PMe rather than PPh for the

3 3

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Research Article

Scheme 3

Scheme 4

Reagents and conditions: (a) TBAF, THF, rt, 0.5 h, 96%; (b) (i)

MsCl, Et N, DCM, 0 °C, 0.5 h, (ii) NaN , DMF, 50 °C, 8 h, 84% over

steps; (c) (i) PMe , THF/H O, rt, 5 h, (ii) Boc O, NaHCO , rt, 2

3 2 2 3

h, 61−63% over 2 steps; (d) Dess−Martin periodinane, CH Cl , 81%;

(

e) NaBH , EtOH, 1:1 mixture of 1, 99%; (f) (i) PPh , DIAD, THF,

Reagents and conditions: (j) 3-azidopropan-1-amine, CuSO

sodium ascorbate, t-BuOH, 80 °C, 3 h; (k) 5-cyclohexylpentanoic

acid 10{1}, EDC·HCl, Et N, DCM, 4 h, 95% over 2 steps from 5S;

(l) TFA, DCM, 0.5 h; (m) (i) 2-biphenylyl isocyanate 12{1}, Et N,

DCM, 16 h, (ii) 6 N HCl, MeOH, 16 h, 85% over 3 steps from

4 2

·5H O,

rt, 0.5 h, (ii) NaOH, MeOH/H O, rt, 5 h, 87% over 2 steps; (g) (i)

MsCl, Et N, DCM, 0 °C, 0.5 h, (ii) NaN , DMF, 50 °C, 8 h, 52% over

steps; (h) DEAD, PPh , THF, 75%; (i) NaN , DMF, 70 °C, 1 h,

9%.

1S{1}; (n) (i) 3,3,3-triphenylpropionic acid 14{1}, EDC·HCl, Et N,

DCM, 4 h, (ii) TFA, DCM, H O, 16 h, 83% over 3 steps from

transformation of 3S to 5R was associated with an increase in

the yield from 52% to 83%.

11S {1 }.

37,38

Chemical Synthesis of Model Molecules. With alkynes

S and 5R in hand, model studies to explore the reaction

conditions necessary to accomplish the practical library

synthesis could be investigated (Scheme 4). 3-Azidopropan-

-amine was conjugated to alkyne 5S using catalytic CuSO₄

and sodium ascorbate via a Cu-catalyzed azide−alkyne

cycloaddition (CuAAC) to give triazole intermediate 9S,

which was directly coupled with 5-cyclohexylpentanoic acid

0{1} (Figure 2) to give 11S{1} in 95% overall yield over two

Figure 2. Set of acids 10{1−2} for diversity.

steps. The N-Boc deprotection of 11S{1} was performed

under acidic TFA conditions to give the corresponding amine

intermediate, which was directly treated with 2-biphenylyl

isocyanate 12{1} to form a urea-based linkage. After global

deprotection using 6 N HCl in MeOH, 13S{1,1} was smoothly

obtained after simple puriﬁcation. Likewise, 13R{1,1}, the C5′

epimer of 13S{1,1}, was similarly prepared from alkyne 5R.

Instead of a, (thio-) urea bond formation, another conjugation

method via an amide bond formation could also be established

with the treatment of mixture of the amine intermediate which

was prepared from N-Boc deprotection of 11S{1}, 3,3,3-

triphenylpropionic acid 14{1}, EDC, and triethylamine. After

simple extraction and global deprotection, 15S{1,1} was also

obtained in 83% yield from 11S{1}.

Library Synthesis. The successful synthesis of 13S{1,1}

and 15S{1,1} from 5S and 13R{1,1} from 5R encouraged us

to attempt the preparation of a more diverse library. To

improve eﬃciency, we sought to use semiautomated equip-

ment. Requisite precursors 11S{1−2} and 11R{1−2} bearing

a lipophilic substituent at R and incorporating conﬁguration

diversity at C5′ were prepared with the assistance of a solution-

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6 acid reagents 14 {1 −16 } (Figure 4 ) were reacted with

Research Article

phase synthesizer, liquid handler, and evaporator as shown in

Scheme 5 (also see in SI ). To simplify our process, the ﬁnal

puriﬁcation was accomplished by solid-phase extraction (SPE).

Scheme 5. General Route for the Preparation of Key

Precursors 11S{1−2} and 11R{1−2} from Alkynes 5S or 5R

A total of 16 isocyanate reagents 12{1−16} (Figure 3) and

Figure 4. Set of acids 14{1−16} for diversity.

Scheme 6. General Procedure for the Semiautomatic

Synthesis of the Desired Library

Figure 3. Set of isocyanates 12{1−16} for diversity

nucleoside-based substrates 11S{1−2} and 11R{1−2} accord-

ing to the protocols described above. Some minor changes

were made to the above protocols, to accommodate the

automated equipment. For example, N-Boc deprotection of the

nucleoside-based substrate 11S{1} under acidic conditions

(

step i) was performed, followed by direct evaporation,

individual treatment with isocyanate reagents 12{1−16}

step ii), and global deprotection (step iii) to obtain desired

(

target molecules 13S{1−2,1−16} and 13R{1−2,1−16} after

simple solid-phase extraction (Scheme 6). Likewise, the amide

bond formation in step ii would also be performed to get the

desired 16 compounds 15S{1,1−16}. Notably, the crude

products were evaporated by speed-vac and washed by

solution-phase extraction assisted by a liquid handler. Most

impurities, derived from the excess of isocyanate or acid

reagents, were easily removed by further solid-phase extraction

with silica gel and 1/1 mixture of ethyl acetate/hexanes as

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Yields after overall synthesis. Purities shown in parentheses.

Research Article

Table 1. Yields and Purities of the Library via a Urea or Amide Bond Formation with Nucleoside-Based Substrates and

Isocyanate or Acid Reagents

product

yield (purity)

product

yield (purity)

product

yield (purity)

product

yield (purity)

product

yield (purity)

13S{1,1}

13S{1,2}

13S{1,3}

13S{1,4}

13S{1,5}

13S{1,6}

13S{1,7}

13S{1,8}

13S{1,9}

13S{1,10}

13S{1,11}

13S{1,12}

13S{1,13}

13S{1,14}

13S{1,15}

13S{1,16}

85 (98)

30 (99)

51 (99)

31 (99)

58 (99)

51 (99)

63 (98)

62 (97)

11 (95)

56 (98)

56 (99)

53 (99)

72 (99)

68 (99)

65 (98)

25 (99)

13S{2,1}

13S{2,2}

13S{2,3}

13S{2,4}

13S{2,5}

13S{2,6}

13S{2,7}

13S{2,8}

13S{2,9}

13S{2,10}

13S{2,11}

13S{2,12}

13S{2,13}

13S{2,14}

13S{2,15}

13S{2,16}

56 (96)

57 (99)

56 (97)

32 (99)

67 (95)

49 (98)

51 (96)

36 (94)

57 (98)

21 (97)

50 (99)

53 (99)

60 (99)

53 (98)

39 (95)

8 (97)

13R{1,1}

13R{1,2}

13R{1,3}

13R{1,4}

13R{1,5}

13R{1,6}

13R{1,7}

13R{1,8}

13R{1,9}

13R{1,10}

13R{1,11}

13R{1,12}

13R{1,13}

13R{1,14}

13R{1,15}

13R{1,16}

41 (94)

23 (99)

62 (97)

70 (96)

64 (95)

45 (98)

76 (96)

50 (97)

31 (93)

40 (98)

37 (97)

45 (98)

49 (95)

29 (99)

27 (85)

62 (99)

13R{2,1}

13R{2,2}

13R{2,3}

13R{2,4}

13R{2,5}

13R{2,6}

13R{2,7}

13R{2,8}

13R{2,9}

13R{2,10}

13R{2,11}

13R{2,12}

13R{2,13}

13R{2,14}

13R{2,15}

13R{2,16}

28 (96)

36 (99)

52 (98)

50 (96)

54 (99)

53 (99)

39 (98)

45 (96)

25 (97)

41 (99)

56 (97)

24 (93)

46 (96)

51 (99)

35 (99)

41 (99)

15S{1,1}

15S{1,2}

15S{1,3}

15S{1,4}

15S{1,5}

15S{1,6}

15S{1,7}

15S{1,8}

15S{1,9}

15S{1,10}

15S{1,11}

15S{1,12}

15S{1,13}

15S{1,14}

15S{1,15}

15S{1,16}

83 (94)

53 (99)

83 (99)

35 (99)

51 (96)

62 (88)

40 (93)

60 (95)

26 (89)

42 (96)

31 (89)

18 (96)

87 (85)

64 (85)

30 (99)

23 (99)

Figure 5. Morphological study of the compounds toward Bacillus subtilis. The bacterial cells (OD₆₀₀_n_m= 0.1−0.2) were incubated with 100 μm

compounds. The images show the eﬀect of the compounds indicated by their code number after 3 h on B. subtilis 168 cells.

eluent, and desired products were subsequently collected by

using pure EtOAc as the eluent.

bacterial cells treated with 13S{1,13} or 13R{1,13} consisted

of long ﬁlament and undivided cells compared with the control

group. Most of the aﬀected bacteria attached to each other via

their poles. These observations indicate that bacterial cell

division is perturbed and cell separation is obstructed by these

compounds. In contrast, compounds 13R{1,7} had no eﬀect

on bacterial shape, despite their structures diﬀering only subtly

from compounds 13S{1,13} and 13R{1,13}. The underlying

mechanisms, conditions, and other factors underlying both

these contrasting results and the changes observed remain to

be explored.

Library members (80 members) were characterized by

NMR, HRMS, HPLC, and optical rotation. Purities ranged

from 85% to 99%, with an average purity of 97% (Table 1).

Yields ranged from 8% to 87% in three steps. The low yields of

some products might be due to the solid-phase extraction in

the unoptimized condition; whereas, their purity was qualiﬁed

for further biological study.

Biological Evaluation of Selected Compounds. With

the uridine-based library in hand, biological studies could

commence. Due to their accessibility, we choose to undertake

39,40

CONCLUSION

antibacterial and bacterial morphological studies.

None of

■

the compounds synthesized were found to exhibit antibacterial

activity against Gram-negative bacterial strains, such as

Acinetobacter baumannii, at a concentration of 100 μm,

presumably because they could not penetrate the bacterial

membranes (data not shown). However, in the morphological

study, several compounds were found to perturb the growth of

Bacillus subtilis (Figure 5). For example, the phenotype of

A structurally diverse, natural product-inspired uridine-

containing library (80 members) incorporating two substituent

diversity positions and one conﬁguration diversity position was

eﬃciently synthesized by elaboration of the key intermediate

5S and 5R (bearing an alkyne group and Boc-N-protected

amino group). Semiautomated equipment including a solution-

phase synthesizer, multichannel liquid handler, and centrifugal

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Research Article

vacuum concentrator were used to speed up the synthesis of

this library, and the rapid puriﬁcation of its members was

accomplished using solid-phase extraction. The conditions

developed in this work are anticipated to enable the generation

of even more structurally diverse, uridine moiety-containing

libraries in the future. A preliminary bacterial morphology

study showed that subtle structural diﬀerences of uridine

compounds lead diﬀerent morphology and shape; a more

extensive bioevaluation of this library (forward chemical

genetic screen) is underway, and the results will be published

in due course.

General Procedure of Model Molecules 11S{1−2} and

11R{1−2}. To a solution of alkyne 5S or 5R (1 equiv) and 3-

azidopropan-1-amine (1.5 equiv) in tert-butanol was succes-

sively added CuSO (0.1 equiv) and sodium ascorbate (0.3

equiv). The mixture was stirred at 80 °C for 3 h and diluted

with EtOAc and water. The aqueous phase was extracted with

EA, and the combined organic layers were dried over MgSO₄,

ﬁltered, and concentrated to get amine. A solution of amine(1

equiv) in DCM was added with acid(1.5 equiv), EDC·HCl

(

1.5 equiv) and triethylamine (3 equiv). After 4 h, the reaction

was quenched and the organic layer was extracted with 1 N

HCl. The combined organic layers were dried over MgSO₄,

ﬁltered and concentrated in vacuum. The residue was puriﬁed

by column chromatography (EtOAc) to get the corresponding

product.

EXPERIMENTAL SECTION

■

General Information and Instrumentation. All chem-

icals were obtained from commercial suppliers and used

without further puriﬁcation. Flash column chromatography

was performed on silica gel of 40−63 μm particle size. NMR

Compound 11S{1}. Yield: 95% over 2 steps. R = 0.2 (EA).

H NMR (600 MHz, MeOD) δ 7.91 (s, 1H), 7.60 (d, 1H, J =

spectra were recorded on dilute solutions in D O, CDCl and

8.0 Hz), 5.70 (br, 1H), 5.66 (d, 1H, J = 8.0 Hz), 5.17 (d, 1H, J

MeOD on Bruker AVANCE 600 spectrometers at ambient

temperature. High-resolution ESI mass spectra were recorded

on a Bruker Daltonics spectrometer. Parallel synthesis was

performed on Mini-Block synthesizer and the reaction vessel

5.0 Hz), 5.11 (d, 1H, J = 5.0 Hz), 4.41 (t, 2H, J = 7.0 Hz),

.36 (br, 1H), 3.19 (t, 2H, J = 7.0 Hz), 2.18 (t, 2H, J = 7.5

Hz), 2.11−2.05 (m, 2H), 1.73−1.63 (m, 5H), 1.60−1.55 (m,

H), 1.52 (s, 3H), 1.42 (s, 9H), 1.35−1.30 (m, 5H), 1.25−

.15 (m, 6H), 0.92−0.85 (m, 2H). C NMR (150 MHz,

(

115 × 12) mm. Multiple-functional liquid handler (Freedom

EVO, TECAN) was utilized for extraction and separation.

Solvent evaporation was performed on Thermo Scientiﬁc Savat

Explorer SpeedVac Concentrator Explorer-220.

MeOD) δ 176.7, 166.4, 157.9, 152.3, 147.5, 145.4, 124.7,

15.7, 103.0, 95.6, 89.1, 85.2, 82.5, 80.7, 50.4, 49.7, 39.0, 38.5,

7.5, 37.3, 34.7 (× 2), 31.3, 28.9 (×3), 27.9, 27.7, 26.6 (×3),

6.4, 25.7. HRMS calculated for [C H N O + H]

Preparation of the Key Intermediate 5S. A mixture of

S (144 mg, 0.356 mmol) and NaN in DMF (0.2 mL, 0.21

74.3872, found 674.3895.

mmol) was stirred at 70 °C for 1.0 h. The reaction residue was

extracted with EtOAc. The organic layers were dried over

Compound 11S{2}. Yield: 97% over 2 steps. R = 0.5 (EA).

H NMR (600 MHz, MeOD) δ 7.91 (s, 1H), 7.60 (d, 1H, J =

MgSO and concentrated. The reaction mixture was puriﬁed

.0 Hz), 5.70 (br, 1H), 5.66 (d, 1H, J = 8.0 Hz), 5.17 (br, 1H),

.11 (d, 1H, J = 4.8 Hz), 4.41 (t, 2H, J = 6.9 Hz), 4.36 (br,

H), 3.19 (t, 2H, J = 6.6 Hz), 2.18 (t, 2H, J = 7.5 Hz), 2.11−

.05 (m, 2H), 1.62−1.57 (m, 2H), 1.52 (s, 3H), 1.42 (s, 9H),

by column chromatography EtOAc/hexane = 1/1) to give 5S

(

110 mg, 0.246 mmol, 69%). R = 0.3 (EtOAc/hexane = 1/1).

H NMR (600 MHz, CD OD) δ 9.78 (brs, 1H), 7.49 (d, 1H, J

8.1 Hz), 5.90 (d, 1H, J = 3.3 Hz), 5.76 (d, 1H, J = 8.1 Hz),

.82 (dd, 1H, J = 6.4, 3.2 Hz), 4.76 (dd, 1H, J = 6.4, 3.2 Hz),

.50 (d, 1H, J = 5.2 Hz), 4.23 (dd, 1H, J = 5.1, 3.2 Hz), 1.57

.34−1.26 (m, 25H), 0.92−0.87 (m, 2H). C NMR (150

MHz, MeOD) δ 176.7, 166.4, 157.9, 152.3, 147.5, 145.4,

124.7, 115.7, 103.0, 95.6, 89.1, 85.2, 82.5, 80.7, 50.4, 49.7, 37.5,

37.3, 33.2, 31.3, 30.9 (× 5), 30.8, 30.6 (× 2), 30.5, 28.9 (× 3),

27.6, 27.2, 25.7, 23.9, 14.6. HRMS calculated for [C H N O

(

s, 3H) 1.33 (s, 3H), 0.98 (t, 9H, J = 7.9 Hz), 0.62 (m, 6H, J =

.9 Hz). C NMR (150 MHz, CD OD) δ 163.4, 150.1, 140.9,

14.8, 102.9, 97.3, 92.9, 92.3, 86.2, 83.7, 80.9, 54.6, 27.0, 25.1,

H] 732.4654, found 732.4677.

.2 (×3), 4.0 (×3). HRMS calcd for [C H NO Si+H]

Compound 11R{1}. Yield: 91% over 2 steps. R = 0.2 (EA).

48.2011, found 448.2013.

H NMR (600 MHz, MeOD) δ 7.89 (s, 1H), 7.52 (d, 1H, J =

Preparation of the Key Intermediate 5R. A mixture of

S (23.0 mg, 0.07 mmol) and 1 M trimethylphosphine in THF

.9 Hz), 5.74 (br, 1H), 5.64 (d, 1H, 7.9 Hz), 5.16 (d, 1H, 6.7

Hz), 5.02 (br, 1H), 4.96 (br, 1H), 4.40 (t, 2H J = 6.8 Hz), 4.32

(

0.2 mL, 0.21 mmol) was added in THF/H O (1/1) and

(

br, 1H), 3.18 (t, 2H J = 6.5 Hz), 2.18 (t, 2H, J = 7.4 Hz),

stirred for 5 h. The reaction mixture was added di-tert-butyl

dicarbonate (0.03 mL, 0.14 mmol) and sodium bicarbonate

.10−2.04 (m, 2H), 1.75−1.63 (m, 5H), 1.60−1.51 (m, 2H),

.53 (s, 3H), 1.45 (s, 9H), 1.35−1.30 (m, 5H), 1.26−1.14 (m,

(

11.6 mg, 0.14 mmol). The reaction mixture was stirred for 2

H), 0.92−0.84 (m, 2H). C NMR (150 MHz, MeOD) δ

76.7, 166.3, 157.8, 152.0, 147.2, 144.7, 124.7, 115.8, 103.0,

4.8, 88.9, 85.2, 82.9, 80.9, 50.2 (×2), 39.0, 38.5, 37.5, 37.3,

h. The reaction residue was extracted with EtOAc. The organic

layers were dried over MgSO and concentrated. The reaction

mixture was added TBAF (1.0 M in THF, 2.4 mL, 2.40 mmol)

and stirred for 0.5 h, then concentrated, puriﬁed by column

chromatography (EtOAc/hexane = 2/1) to give 5R (13.5 mg,

4.7 (×2), 31.3, 28.8 (×3), 28.0, 27.7, 27.6 (×3), 27.4, 25.7.

HRMS calculated for [C H N O + H] 674.3872, found

33 51

674.3909.

.044 mmol, 63% over 3 steps). R = 0.7 (EtOAc/hexane = 2/

Compound 11R{2}. Yield: 73% over 2 steps. R = 0.5 (EA).

). H NMR (600 MHz, DMSO-d ) δ 11.4 (brs, 1H), 7.67 (d,

H NMR (600 MHz, MeOD) δ 7.91 (s, 1H), 7.60 (d, 1H, J =

8.0 Hz), 5.70 (br, 1H), 5.66 (d, 1H, J = 8.0 Hz), 5.17 (br, 1H),

5.11 (d, 1H, J = 4.8 Hz), 4.41 (t, 2H, J = 6.9 Hz), 4.36 (br,

1H), 3.19 (t, 2H, J = 6.6 Hz), 2.18 (t, 2H, J = 7.5 Hz), 2.11−

2.05 (m, 2H), 1.62−1.57 (m, 2H), 1.52 (s, 3H), 1.42 (s, 9H),

H, J = 8.0 Hz), 7.30 (d, 1H, J = 9.1 Hz), 5.73 (d, 1H, J = 1.4

Hz), 5.57 (d, 1H, J = 8.0 Hz), 5.15 (d, 1H, J = 6.1 Hz), 4.87

(

dd, 1H, J = 6.3, 3.0 Hz), 4.58 (m, 1H, J = 9.1, 7.7 Hz), 4.02

dd, 1H, J = 9.1, 3.0 Hz), 3.30 (s, 3H), 1.46 (s, 3H), 1.32 (s, 9

H), 1.29 (s, 3H). C NMR (150 MHz, DMSO-d ) δ 163.2,

55.0, 150.2, 142.8, 113.2, 101.8, 92.5, 86.7, 83.2, 81.6, 81.4,

1.34−1.26 (m, 25H), 0.92−0.87 (m, 2H). C NMR (150

MHz, MeOD) δ 176.7, 166.4, 157.9, 152.3, 147.5, 145.4,

124.7, 115.7, 103.0, 95.6, 89.1, 85.2, 82.5, 80.7, 50.4, 49.7, 37.5,

8.7, 74.5, 43.9, 28.1 (×3), 26.8, 25.0. HRMS calculated for

[C H N O + H] 430.1585, found 430.1565.

ACS Comb. Sci. XXXX, XXX, XXX−XXX

ACS Combinatorial Science

Schinazi, R. F. Metabolism, biochemical actions, and chemical

Research Article

7.3, 33.2, 31.3, 30.9 (×5), 30.8, 30.6 (×2), 30.5, 28.9 (×3),

(6) Shelton, J.; Lu, X.; Hollenbaugh, J. A.; Cho, J. H.; Amblard, F.;

synthesis of anticancer nucleosides, nucleotides, and base analogs.

Chem. Rev. 2016, 116, 14379−14455.

7.6, 27.2, 25.7, 23.9, 14.6. HRMS calculated for [C H N O

H] 732.4654, found 732.4677.

7) Zhao, C.; Huang, T.; Chen, W.; Deng, Z. Enhancement of the

ASSOCIATED CONTENT

■

diversity of polyoxins by a thymine-7-hydroxylase homolog outside

the polyoxin biosynthesis gene cluster. Appl. Environ. Microbiol. 2010,

76, 7343−7347.

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acscombsci.0c00011 .

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AUTHOR INFORMATION

Corresponding Authors

■

Wei-Chieh Cheng − Genomics Research Center, Academia

Sinica, Taipei 115, Taiwan; Department of Chemistry,

National Cheng-Kung University, Tainan 701, Taiwan;

Department of Applied Chemistry, National Chiayi University,

Chiayi 600, Taiwan; Department of Medicinal and Applied

Chemistry, Kaohsiung Medical University, Kaohsiung 807,

Taiwan; orcid.org/0000-0002-9319-3010;

Email: wccheng6l09@gmail.com

Lee-Chiang Lo − Department of Chemistry, National Taiwan

University, Taipei 106, Taiwan; orcid.org/0000-0002-

10) Mitachi, K.; Aleiwi, B. A.; Schneider, C. M.; Siricilla, S.; Kurosu,

M. Stereocontrolled total synthesis of muraymycin D1 having a dual

mode of action against mycobacterium tuberculosis. J. Am. Chem. Soc.

2016, 138, 12975−12980.

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phosphoglycosyl transferases: initiators of glycan biosynthesis at the

membrane interface. Glycobiology 2017, 27, 820−833.

(12) McDonald, L. A.; Barbieri, L. R.; Carter, G. T.; Lenoy, E.;

Lotvin, J.; Petersen, P. J.; Siegel, M. M.; Singh, G.; Williamson, R. T.

Structures of the muraymycins, novel peptidoglycan biosynthesis

inhibitors. J. Am. Chem. Soc. 2002, 124, 10260 −10261.

821-1690; Email: lclo@ntu.edu.tw

(13) Takatsuki, A.; Arima, K.; Tamura, G. Tunicamycin, a new

antibiotic. I. Isolation and characterization of tunicamycin. J. Antibiot.

971, 24, 215−223.

14) Takatsuki, A.; Kawamura, K.; Okina, M.; Kodama, Y.; Ito, T.;

Tamura, G. The structure of tunicamycin. Agric. Biol. Chem. 1977, 41,

307−2309.

15) Moukha-Chafiq, O.; Reynolds, R. C. Parallel solution-phase

Authors

(

Wan-Ju Liu − Genomics Research Center, Academia Sinica,

Taipei 115, Taiwan; Department of Chemistry, National

Taiwan University, Taipei 106, Taiwan

Kung-Hsiang Hu − Genomics Research Center, Academia

Sinica, Taipei 115, Taiwan

synthesis and general biological activity of a uridine antibiotic analog

library. ACS Comb. Sci. 2014 , 16 , 232 −237.

Yee-Ling Tan − Genomics Research Center, Academia Sinica,

Taipei 115, Taiwan

Yan-Ting Lin − Genomics Research Center, Academia Sinica,

Taipei 115, Taiwan

Wei-An Chen − Genomics Research Center, Academia Sinica,

Taipei 115, Taiwan

(16) Sun, D.; Lee, R. E. Solid-phase synthesis of a thymidinyl

dipeptide urea library. J. Comb. Chem. 2007 , 9 , 370 −385.

(17) Park, J. W.; Nam, S. J.; Yoon, Y. J. Enabling techniques in the

search for new antibiotics: combinatorial biosynthesis of sugar-

containing antibiotics. Biochem. Pharmacol. 2017, 134, 56−73.

(18) Cheng, W.-C.; Guo, C.-W.; Lin, C.-K.; Jiang, Y.-R Synthesis

Complete contact information is available at:

https://pubs.acs.org/10.1021/acscombsci.0c00011

and inhibition study of bicyclic iminosugar-based alkaloids, scaffolds,

and libraries towards glucosidase. Isr. J. Chem. 2015, 55, 403−411.

(19) Cheng, W.-C.; Lin, C.-K.; Li, H.-Y.; Chang, Y.-C.; Lu, S.-J.;

Notes

Chen, Y.-S.; Chang, S.-Y A combinatorial approach towards the

synthesis of non-hydrolysable triazole-iduronic acid hybrid inhibitors

of human α -l-iduronidase: discovery of enzyme stabilizers for the

potential treatment of MPSI. Chem. Commun. 2018, 54, 2647−2650.

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

(

20) Shih, H.-W.; Chen, K.-T.; Chen, S.-K.; Huang, C.-Y.; Cheng,

We thank Academia Sinica and the Ministry of Science and

Technology for ﬁnancial support.

T.-J. R.; Ma, C.; Wong, C.-H.; Cheng, W.-C Combinatorial approach

toward synthesis of small molecule libraries as bacterial trans-

glycosylase inhibitors. Org. Biomol. Chem. 2010, 8, 2586−2593.

(21) Chen, K.-T.; Huang, D.-Y.; Chiu, C.-H.; Lin, W.-W.; Liang, P.-

H.; Cheng, W.-C. Synthesis of diverse N-substituted muramyl

dipeptide derivatives and their use in a study of human NOD2

stimulation activity. Chem. - Eur. J. 2015, 21, 11984−11988.

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